This invention relates to the use of arachadonic acid for achieving enhanced cultures of fish larvae and broodstock.
The common practice of transferring fish larvae from controlled hatchery environment to less controlled grow-out systems generally occurs near larval metamorphosis. During this stage of development, larvae are physiologically stressed and the immune response is weak. As a result, heavy mortality frequently occurs. In addition, stressful conditions such as crowding, nutritional deficiencies, and heavy organic and/or metal loads are potent suppressors of the immune system (Mazur and Iwama 1993; Pickering and Pottinger 1989).
The grow-out culture environment can also harbor opportunistic and pathogenic bacteria, which can infect the stressed larvae and reduce growth and survivorship (Andrews and Harris 1986; Vadstein 1996). Many aspects of the stress response and immune function can be modulated by nutritional factors, including vitamins, proteins, lipids, and minerals (reviewed by Chandra 1988; Johnston 1985). Among the most common micro-supplements, antioxidant-vitamins such as C and F appear to increase disease resistance and boost the fish immune system (Blazer 1992; Hardie et al. 1991; Gapasin et al. 1998). Other substances such as yeast glucan (Jeney and Anderson 1993b) and Levamnisole or mannuronic rich alginate (Anderson 1992; Skjermo et al. 1995) are also effective in stimulating the non-specific immune system in fish. Furthermore, numerous studies, mostly in mammals, have suggested that dietary long chain polyunsaturated fatty acids (PUFA) have a well marked influence on the function of both the stress and immune systems (Calder 1998; Galli and Marangoni 1997; Harbige 1998; Mills et al. 1994). In fish however, and especially in their larval stages, the dietary PUFA effect on the stress and the immune systems are less well known (partially reviewed in Kanazawa 1997).
Stress and non-specific immune responses have been demonstrated in fish from early age (Fletcher 1997). Fish larvae, however, do not have a specific immunity that is as well developed as the adult (Mughal and Manning 1986; Ruglys 1985). Fish larvae therefore are dependent on the non-specific immune system as the major line of defense against microorganisms (Ellis 1988). Non-specific protective immunity can be demonstrated in fish larvae as early as 14-16 days post hatch (Botharn and Manning 1981; Tatner and Home 1983). It is believed that the main cellular defense in fish larvae is by phagocytosis, a process that is not as well characterized as other responses such as the inflammatory function of neutrophils and monocytes in the larvae. Stress conditions can depress the immune-function, eliciting neuroendocrine responses along the hypothalamus-pituitary-interrenal (HPI) axis, and resulting in increased levels of catecholamines and glucocorticoid hormones, which in turn induce a wide variety of metabolic and osmotic changes including immunosuppressive effects (Balm 1997; Barton and Iwama 1991).
Diets rich in PUFA are often associated with suppression of the immune system (Kiron et al. 1995; Thompson et al. 1996), but the mechanism for such suppression is not clear. Animal studies have indicated that these fatty acids are cellular targets for oxygen radicals, which break the fatty acids down into several toxic carbonyl compounds. The carbonyl compounds in turn initiate intracellular formation of reactive oxygen species (ROS) and lipid peroxidation products (Maziere et al. 1999).
Changes in dietary levels and the ratio of n-3 and n-6 fatty acids can modulate the production of bioactive lipids, thereby affecting stress and pathogen resistance (Calder et al. 1990; Kiron et al. 1995; Kraul et al. 1993; Palmblad 1987; Tort et al. 1996). Cell enrichment with n-3 and n-6 fatty acids may also affect the immune system through the production of eicosanoids and cytokines (Harbige 1998; Khalfoun et al. 1997), and by reducing lymphocyte proliferation, and monocyte and neutrophil chemotaxis (Ainsworth et al. 1991; Calder 1998; Pickering and Pottinger 1987, respectively).
The n-6 fatty acids, in particular arachidonic acid (AA), play a central role in the production of eicosanoids peroxidation products, as well as initiating the production of ROS. Furthermore, stress stimuli such as free radicals and high osmotic loads induce stress-activated protein kinases (SAPKs) in a wide variety of cells. The induction of SAPKs in turn primes cytosolic phospholipase A2 (cPLA2) to release AA from tissue phospholipids (Buschbeck et al. 1999; Maziere et al. 1999). On the other hand, n-3 PUFAs inhibit the metabolism of n-6 PUFA, thereby promoting a shift toward the formation of less reactive eicosaniods, and diminished superoxide formation (Palombo et al. 1999). Considering the common practice in many hatcheries to feed fish larvae with highly enriched n-3 PUFA diets, the possibility exists that extensive exposure to n-3 PUFA rich lipids may eventually suppress the larval capacity to cope with stressful events and to develop an appropriate non-specific immune response.
Considering nutritional requirements of illustrative specific fish species, the white bass Morone chrysops is a freshwater fish species, closely related to the striped bass Morone saxatilis. Adults are piscivorus, occupying freshwater habitats and as such may retain and preferentially conserve their limited dietary n-3 HUFA sources. The Morone larvae, in common with many other commercially important marine larval species, are not able to elongate and desaturate n-3 and n-6 precursors into their HUFA metabolites. In fact, of the four marine teleosts including ayu Plecoglossus altivelis, red sea bream Pagrus major and globefish fugu rubripes rubripes, members of this genus demonstrate the lowest conversion rate of C-18:n-3 precursor to its C-20 and C-22 fatty acids metabolites. Thus, larvae must be provided with sufficient levels of HUFA, in order to meet the nutritional requirements for optimal growth.
In light of the known competition between n-6 and n-3 fatty acids series for their common enzymes, the relationship between fatty acid composition in larval body tissue and dietary supplementation of n-6 and n-3 fatty acids is of interest. In fish and mammal brain tissue and eye retinal tissue, docosahexaenoic acid (DHA, 22:6n-3) is the most prominent fatty acid. It has been observed that neuronal differentiation coincides with rapid DHA accumulation in structural phospholipids of the central nervous system. In addition to DHA, arachidonic acid (AA, 20:4n-6) is also a critical component of membrane lipids and is specifically accumulated in brain phospholipids during early development. Arachidonic acid plays an active role in signal transduction both through the production of eicosanoids in whole body tissues, and as a second messenger in neural tissue. Recent studies have shown that dietary supplementation of AA together with DHA inhibited DHA accretion in the phospholipids fraction of tissue lipids. This antagonistic relationship is potentially detrimental to the proper function of brain and neural tissues, where DHA is believed to serve a critical function.
The adverse effects on larval growth and survival because of excessive essential fatty acids (EFAs) in the diet have been previously reported. However, in spite of efforts to establish absolute requirements for AA for some fish species, the combined requirements of AA and DHA, in both absolute and relative amounts, are not known for any species.
As another aspect of aquaculture nutrition relevant to the present invention, fish meal and fish oil currently are the main ingredients in finfish and marine shrimp nutrition. Together they provide a good balance of protein (amino acids) and lipids (long chain n-3 highly unsaturated fatty acids) in a highly digestible energy-dense form. Studies have shown that diets containing fish-based ingredients generally perform better in terms of growth and feed efficiency than diets containing alternative plant based sources. However, as a result of a decreasing supply of fishery byproducts and concomitant concerns about the quality of such byproducts, the aquaculture industry is actively investigating alternative nutrient sources.
The foregoing discussion highlights the continuing need for improved nutritional source materials in the aquaculture industry.